scholarly journals Biosynthesis of high density lipoprotein by chicken liver: intracellular transport and proteolytic processing of nascent apolipoprotein A-1.

1985 ◽  
Vol 101 (4) ◽  
pp. 1219-1226 ◽  
Author(s):  
D Banerjee ◽  
T K Mukherjee ◽  
C M Redman
Keyword(s):  
Amino Acids ◽  
Sequence Analysis ◽  
Golgi Apparatus ◽  
Intracellular Transport ◽  
Proteolytic Processing ◽  
Chicken Liver ◽  
Golgi Cell ◽  
Apolipoprotein A ◽  
Cell Fractions

To study the in vivo processing and secretion of Apolipoprotein A-I (Apo A-I), young chickens were administered individual L-[3H]amino acids intravenously and the time of intracellular transport of nascent Apo A-I from rough endoplasmic reticulum (RER) to the Golgi apparatus was measured. Within 3 to 9 min there was maximal incorporation of radioactivity into Apo A-I in both the RER and the Golgi cell fractions. By contrast, the majority of radioactive albumin was also present in the RER by 3 to 9 min, but did not reach peak amounts in the Golgi fraction until 9 to 25 min. Both radioactive Apo A-I and albumin appeared in the blood at about the same time (between 20 and 30 min). NH2-terminal amino acid sequence analysis of nascent intracellular Apo A-I showed that it contains a pro-hexapeptide extension identical to that of human Apo A-I. After 30 min of administration of radioactive amino acids radioactive Apo A-I was isolated by immunoprecipitation from the liver and serum. NH2-terminal sequence analysis of 20 amino acids indicated that chicken liver contained an equal mixture of nascent pro-Apo A-I and fully processed Apo A-I, whereas the serum only contained processed Apo A-I. Further studies showed that the RER only contained pro-Apo A-I, whereas a mixture of pro-Apo A-I and processed Apo A-I was found in the Golgi complex. These results indicate that, in chicken hepatocytes, there is a more rapid transport of Apo A-I than of albumin from the RER to the Golgi cell fractions, and that Apo A-I remains in the Golgi apparatus for a longer period of time before it is secreted into the blood. In addition these studies show that the in vivo proteolytic processing of chicken pro-Apo A-I to Apo A-I occurs in the Golgi cell fractions.

scholarly journals Deletion of the propeptide of apolipoprotein A-I impairs exit of nascent apolipoprotein A-I from the endoplasmic reticulum

1994 ◽  
Vol 302 (3) ◽  
pp. 641-648 ◽  
Author(s):  
R S McLeod ◽  
C Robbins ◽  
A Burns ◽  
Z Yao ◽  
P H Pritchard
Keyword(s):  
Amino Acids ◽  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Intracellular Transport ◽  
Secretory Pathway ◽  
Wild Type ◽  
Mature Form ◽  
Human Apolipoprotein ◽  
Apolipoprotein A

Human apolipoprotein (apo) A-I is secreted as a proprotein of 249 amino acids and is processed extracellularly to the mature form (243 amino acids) by removal of a six-residue propeptide segment. We have examined the role of the apoA-I propeptide in intracellular transport and secretion using transfected baby hamster kidney cells that secreted either proapoA-I (from the wild-type cDNA, A-Iwt) or mature-form apoA-I (from A-I delta pro, a cDNA in which the propeptide sequence was deleted). Deletion of the propeptide from the apoA-I sequence did not affect the rate of apoA-I synthesis, nor did it affect the fidelity of proteolytic removal of the prepeptide. However, the propeptide deletion caused mature-form apoA-I to accumulate within the cells as determined by pulse-chase experiments; the intracellular retention times for the mature-form apoA-I in which the propeptide was prematurely removed was three times longer than that of proapoA-I (t1/2 > 3 h compared with approximately 50 min). There was no detectable degradation of either form of newly synthesized apoA-I. Immunofluorescence microscopy revealed that, whereas the proapoA-I was located predominantly in the Golgi apparatus, large quantities of the mature-form apoA-I were detected in the endoplasmic reticulum and very little was in the Golgi apparatus of A-I delta pro-transfected cells. These findings suggest that the propeptide sequence may be involved in the intracellular transport of apoA-I from the endoplasmic reticulum to the Golgi apparatus. We propose that the function of the propeptide sequence is to facilitate efficient transport of apoA-I through the secretory pathway.

scholarly journals Primary structure requirements for correct sorting of the yeast mitochondrial protein ADH III to the yeast mitochondrial matrix space.

1987 ◽  
Vol 7 (1) ◽  
pp. 294-304 ◽  
Author(s):  
D Pilgrim ◽  
E T Young
Keyword(s):  
Amino Acids ◽  
Enzyme Activity ◽  
Amino Acid ◽  
Proteolytic Processing ◽  
Mitochondrial Matrix ◽  
Wild Type ◽  
Mature Form ◽  
Amino Terminal ◽  
The Matrix

Alcohol dehydrogenase isoenzyme III (ADH III) in Saccharomyces cerevisiae, the product of the ADH3 gene, is located in the mitochondrial matrix. The ADH III protein was synthesized as a larger precursor in vitro when the gene was transcribed with the SP6 promoter and translated with a reticulocyte lysate. A precursor of the same size was detected when radioactively pulse-labeled proteins were immunoprecipitated with anti-ADH antibody. This precursor was rapidly processed to the mature form in vivo with a half-time of less than 3 min. The processing was blocked if the mitochondria were uncoupled with carbonyl cyanide m-chlorophenylhydrazone. Mutant enzymes in which only the amino-terminal 14 or 16 amino acids of the presequence were retained were correctly targeted and imported into the matrix. A mutant enzyme that was missing the amino-terminal 17 amino acids of the presequence produced an active enzyme, but the majority of the enzyme activity remained in the cytoplasmic compartment on cellular fractionation. Random amino acid changes were produced in the wild-type presequence by bisulfite mutagenesis of the ADH3 gene. The resulting ADH III protein was targeted to the mitochondria and imported into the matrix in all of the mutants tested, as judged by enzyme activity. Mutants containing amino acid changes in the carboxyl-proximal half of the ADH3 presequence were imported and processed to the mature form at a slower rate than the wild type, as judged by pulse-chase studies in vivo. The unprocessed precursor appeared to be unstable in vivo. It was concluded that only a small portion of the presequence contains the necessary information for correct targeting and import. Furthermore, the information for correct proteolytic processing of the presequence appears to be distinct from the targeting information and may involve secondary structure information in the presequence.

scholarly journals CONCOMITANT SYNTHESIS OF MEMBRANE PROTEIN AND EXPORTABLE PROTEIN OF THE SECRETORY GRANULE IN RAT PAROTID GLAND

1971 ◽  
Vol 50 (1) ◽  
pp. 187-200 ◽  
Author(s):  
Abraham Amsterdam ◽  
Michael Schramm ◽  
Itzhak Ohad ◽  
Yoram Salomon ◽  
Zvi Selinger
Keyword(s):  
Amino Acids ◽  
Secretory Granule ◽  
De Novo ◽  
Secretory Granules ◽  
Specific Radioactivity ◽  
Staining Intensity ◽  
Granule Membrane ◽  
Rat Parotid ◽  
Cell Fractions

After enzyme secretion the membrane of the secretory granule, which had been fused to the cell membrane, was resorbed into the cell. Experiments were therefore carried out to test whether formation of new secretory granules involves reutilization of the resorbed membrane or synthesis of a new membrane, de novo, from amino acids. Incorporation of amino acids-14C into proteins of various cell fractions was measured in vivo, 30, 120, and. 300 min after labeling. At all times the specific radioactivity of the secretory granule membrane was about equal to that of the granule's exportable content. At 120 and 300 min the specific radioactivity of the granule membrane and of the granule content was much higher than that of any other subcellular fraction. It is therefore concluded that the protein of the membrane is synthesized de novo concomitantly with the exportable protein. The proteins of the granule membrane could be distinguished from those of the granule content by gel electrophoresis. All major bands were labeled proportionately to their staining intensity. The amino acid composition of the secretory granule membrane was markedly different from that of the granule's content and also from that of the mitochondrial membrane. The granule membrane showed a high proline content, 30 moles/100 moles amino acids. The analyses show that the radioactivity of the granule membrane is indeed inherent in its proteins and is not due to contamination by other fractions. The possibility is considered that the exportable protein leaves the endoplasmic reticulum already enveloped by the newly synthesized membrane.

scholarly journals The propeptide of preprosomatostatin mediates intracellular transport and secretion of alpha-globin from mammalian cells.

1989 ◽  
Vol 108 (5) ◽  
pp. 1647-1655 ◽  
Author(s):  
T J Stoller ◽  
D Shields
Keyword(s):  
Amino Acids ◽  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Signal Peptide ◽  
Mammalian Cells ◽  
Intracellular Transport ◽  
Secretory Pathway ◽  
Gh3 Cells ◽  
Alpha Globin ◽  
Regulated Secretory Pathway

We have investigated the role of the somatostatin propeptide in mediating intracellular transport and sorting to the regulated secretory pathway. Using a retroviral expression vector, two fusion proteins were expressed in rat pituitary (GH3) cells: a control protein consisting of the beta-lactamase signal peptide fused to chimpanzee alpha-globin (142 amino acids); and a chimera of the somatostatin signal peptide and proregion (82 amino acids) fused to alpha-globin. Control globin was translocated into the endoplasmic reticulum as determined by accurate cleavage of its signal peptide; however, alpha-globin was not secreted but was rapidly and quantitatively degraded intracellularly with a t 1/2 of 4-5 min. Globin degradation was insensitive to chloroquine, a drug which inhibits lysosomal proteases, but was inhibited at 16 degrees C suggesting proteolysis occurred during transport to the cis-Golgi apparatus. In contrast to the control globin, approximately 30% of the somatostatin propeptide-globin fusion protein was transported to the distal elements of the Golgi apparatus where it was endoproteolytically processed. Processing of the chimera occurred in an acidic intracellular compartment since cleavage was inhibited by 25 microM chloroquine. 60% of the transported chimera was cleaved at the Arg-Lys processing site in native prosomatostatin yielding "mature" alpha-globin. Most significantly, approximately 50% of processed alpha-globin was sorted to the regulated pathway and secreted in response to 8-Br-cAMP. We conclude that the somatostatin propeptide mediated transport of alpha-globin from the endoplasmic reticulum to the trans-Golgi network by protecting molecules from degradation and in addition, facilitated packaging of alpha-globin into vesicles whose secretion was stimulated by cAMP.

scholarly journals The Proinsulin C-peptide—A Multirole Model

2004 ◽  
Vol 5 (1) ◽  
pp. 7-14 ◽  
Author(s):  
Donald F. Steiner
Keyword(s):  
Intracellular Transport ◽  
Secretory Granules ◽  
Proteolytic Processing ◽  
Biologically Active ◽  
Insulin Biosynthesis ◽  
Peptide Structure ◽  
C Peptide ◽  
Additional Role ◽  
Independent Indicator

The C-peptide links the insulin A and B chains in proinsulin, providing thereby a means to promote their efficient folding and assembly in the endoplasmic reticulum during insulin biosynthesis. It then facilitates the intracellular transport, sorting, and proteolytic processing of proinsulin into biologically active insulin in the maturing secretory granules of theβcells. These manifold functions impose significant constraints on the C-peptide structure that are conserved in evolution. After cleavage of proinsulin, the intact C-peptide is stored with insulin in the soluble phase of the secretory granules and is subsequently released in equimolar amounts with insulin, providing a useful independent indicator of insulin secretion. This brief review highlights many aspects of its roles in biosynthesis, as a prelude to consideration of its possible additional role(s) as a physiologically active peptide after its release with insulin into the circulation in vivo.

scholarly journals EVIDENCE FOR THE PARTICIPATION OF THE GOLGI APPARATUS IN THE INTRACELLULAR TRANSPORT OF NASCENT ALBUMIN IN THE LIVER CELL

1970 ◽  
Vol 47 (3) ◽  
pp. 555-567 ◽  
Author(s):  
Hans Glaumann ◽  
Jan L. E. Ericsson
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Liver Cell ◽  
Intracellular Transport ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Parenchymal Liver Cell ◽  
Parenchymal Liver ◽  
Membrane Bound

A comparative biochemical and radioautographic in vivo study was performed to identify the site of synthesis and route of migration of albumin in the parenchymal liver cell after labeling with leucine-14C or leucine-3H via the portal vein. Free cytoplasmic ribosomes, membrane-bound ribosomes, rough- and smooth-surfaced microsomes, and Golgi membranes were isolated. The purity of the Golgi fraction was examined morphologically and biochemically. After administration of leucine-14C, labeled albumin was extracted, and the sequence of transport was followed from one fraction to the other. Approximately 2 min after the intravenous injection, bound ribosomes displayed a maximal rate of leucine-14C incorporation into albumin. 4 min later, a peak was reached for rough microsomes. Corresponding maximal activities for smooth microsomes were recorded at 15 min, and for the Golgi apparatus at ∼20 min. The relative amount of albumin, calculated on a membrane protein basis, was higher in the Golgi fraction than in the microsomes. By radioautography the silver grains were preferentially localized over the rough-surfaced endoplasmic reticulum at the 5 min interval. Apparent activity in the Golgi zone was noted 9 min after the injection; at 15 and 20 min, the majority of the grains were found in this location. Many of the grains associated with the Golgi apparatus were located over Golgi vacuoles containing 300–800 A electron-opaque bodies. It is concluded that albumin is synthesized on bound ribosomes, subsequently is transferred to the cavities of rough-surfaced endoplasmic reticulum, and then undergoes migration to the smooth-surfaced endoplasmic reticulum and the Golgi apparatus. In the latter organelle, albumin can be expected to be segregated together with very low density lipoprotein in vacuoles known to move toward the sinusoidal portion of the cell and release their content to the blood.

A possible role for stable microtubules in intracellular transport from the endoplasmic reticulum to the Golgi apparatus

1994 ◽  
Vol 107 (5) ◽  
pp. 1321-1331 ◽  
Author(s):  
M. Mizuno ◽  
S.J. Singer
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Transfer Process ◽  
Intracellular Transport ◽  
Early Stage ◽  
Critical Role ◽  
Treatment Protocol ◽  
Secretory Proteins ◽  
Random Diffusion

The intracellular transport of secretory proteins involves at an early stage the formation of vesicles from transitional elements of the endoplasmic reticulum (ER) containing these proteins and the transfer of these vesicles to the cis-face of the Golgi apparatus. We propose that the latter transfer process does not occur by random diffusion, but is instead mediated by tracking along stable microtubules. To test this proposal, we have carried out double immunoelectron microscopic labeling experiments on frozen sections of HepG2 hepatoma cells secreting the protein human serum albumin (HSA). By a cycloheximide treatment protocol, the stage during which the transfer of newly synthesized HSA from the ER to the Golgi apparatus occurs in vivo was determined. Sections of the cells were then double immunolabeled using primary antibodies to HSA and to glu-tubulin, the latter specifically detecting stable microtubules. We observed a significantly high frequency of HSA-containing structures between the ER and the Golgi apparatus with which stable microtubules were closely associated. These results support the proposal that stable microtubules may play a critical role in directing the transfer process from the ER to the Golgi apparatus.

scholarly journals Import of rat ornithine transcarbamylase precursor into mitochondria: two-step processing of the leader peptide.

1987 ◽  
Vol 105 (6) ◽  
pp. 2631-2639 ◽  
Author(s):  
E S Sztul ◽  
J P Hendrick ◽  
J P Kraus ◽  
D Wall ◽  
F Kalousek ◽  
...  
Keyword(s):  
Amino Acids ◽  
Cleavage Site ◽  
Liver Mitochondria ◽  
Essential Elements ◽  
Ornithine Transcarbamylase ◽  
Proteolytic Processing ◽  
Leader Peptide ◽  
Rat Liver Mitochondria

The mitochondrial matrix enzyme ornithine transcarbamylase (OTC) is synthesized on cytoplasmic polyribosomes as a precursor (pOTC) with an NH2-terminal extension of 32 amino acids. We report here that rat pOTC synthesized in vitro is internalized and cleaved by isolated rat liver mitochondria in two, temporally separate steps. In the first step, which is dependent upon an intact mitochondrial membrane potential, pOTC is translocated into mitochondria and cleaved by a matrix protease to a product designated iOTC, intermediate in size between pOTC and mature OTC. This product is in a trypsin-protected mitochondrial location. The same intermediate-sized OTC is produced in vivo in frog oocytes injected with in vitro-synthesized pOTC. The proteolytic processing of pOTC to iOTC involves the removal of 24 amino acids from the NH2 terminus of the precursor and utilizes a cleavage site two residues away from a critical arginine residue at position 23. In a second cleavage step, also catalyzed by a matrix protease, iOTC is converted to mature OTC by removal of the remaining eight residues of leader sequence. To define the critical regions in the OTC leader peptide required for these events, we have synthesized OTC precursors with alterations in the leader. Substitution of either an acidic (aspartate) or a "helix-breaking" (glycine) amino acid residue for arginine 23 of the leader inhibits formation of both iOTC and OTC, without affecting translocation. These mutant precursors are cleaved at an otherwise cryptic cleavage site between residues 16 and 17 of the leader. Interestingly, this cleavage occurs at a site two residues away from an arginine at position 15. The data indicate that conversion of pOTC to mature OTC proceeds via the formation of a third discrete species: an intermediate-sized OTC. The data suggest further that, in the rat pOTC leader, the essential elements required for translocation differ from those necessary for correct cleavage to either iOTC or mature OTC.

scholarly journals Biosynthesis of high density lipoprotein by chicken liver: conjugation of nascent lipids with apoprotein A1.

1984 ◽  
Vol 99 (6) ◽  
pp. 1917-1926 ◽  
Author(s):  
D Banerjee ◽  
C M Redman
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Smooth Endoplasmic Reticulum ◽  
Lipid Synthesis ◽  
Density Lipoprotein ◽  
Plasma Lipoproteins ◽  
Golgi Cell ◽  
Intracellular Organelles ◽  
Apoprotein A1 ◽  
Cell Fractions

To study the assembly of newly synthesized lipids with apoprotein A1, we administered [2-3H]glycerol to young chickens and determined the hepatic intracellular sites of lipid synthesis and association of nascent lipids with apoprotein A1. [2-3H]glycerol was rapidly incorporated into hepatic lipids, reaching maximal levels at 5 min, and this preceded the appearance of lipid radioactivity in the plasma. The liver was fractionated into rough and smooth endoplasmic reticulum and Golgi cell fractions. The isolated cell fractions were further subfractionated into membrane and soluble (content) fractions by treatment with 0.1 M Na2CO3, pH 11.3. At various times, the lipid radioactivity was measured in each of the intracellular organelles, in immunoprecipitable apoprotein A1, and in materials that floated at buoyant densities similar to those of plasma lipoproteins. Maximal incorporation occurred at 1 min in the rough endoplasmic reticulum, at 3-5 min in the smooth endoplasmic reticulum, and at 5 min in the Golgi cell fractions. The majority (66-93%) of radioactive glycerol was incorporated into triglycerides with smaller (4-27%) amounts into phospholipids. About 80% of the lipid radioactivity in the endoplasmic reticulum and 70% of that in the Golgi cell fractions was in the membranes. The radioactive lipids in the content subfraction were distributed in various density classes with most nascent lipids floating at a density less than or equal to 1.063 g/ml. Apoprotein A1 from the Golgi apparatus, obtained by immunoprecipitation, contained sixfold more nascent lipids than did that from the endoplasmic reticulum. These data indicate that [2-3H]glycerol is quickly incorporated into lipids of the endoplasmic reticulum and the Golgi cell fractions, that most of the nascent lipids are conjugated with apoproteins A1 in the Golgi apparatus, and that very little association of nascent lipid to apoprotein A1 occurs in the endoplasmic reticulum.

Effects of hypoxia on in vivo glycine-C14 incorporation into pancreatic cell proteins

1965 ◽  
Vol 208 (6) ◽  
pp. 1177-1182 ◽  
Author(s):  
M. Don Turner ◽  
Anne C. Turner
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Liquid Scintillation ◽  
Experimental Series ◽  
Male Rats ◽  
Supernatant Fraction ◽  
Zymogen Granule ◽  
Pancreatic Cell ◽  
Cell Fractions

The effects of graded hypoxia on glycine-C14 incorporation into subcellular components were measured in the intact mammal. Groups of three fasted male rats were injected intraperitoneally with 5 µc of glycine-2-C14 and sacrificed at 5 (or 10), 15, 20, 30, and 45 min by decapitation. In one experimental series the environmental pO2 was maintained at 35 mm Hg for 2 hr before injection and throughout the experiment. In three other experimental series, the pO2 in the sealed chamber was maintained at 58, 48, and 38 mm Hg for 45 min before injection and for the duration of the experiment. The whole pancreases were rapidly removed, cooled, homogenized, and pooled before separation of cell fractions by ultracentrifugation. The specific activities (counts/min per µg of amino acid or protein N) were obtained for plasma and supernatant fraction ("cell sap") amino acids and for the purified proteins of zymogen granule, mitochondrial, microsomal, and cell sap fractions from micro-Kjeldahl analyses and liquid-scintillation counting. No detectable changes were found in the turnover of plasma amino acids during graded hypoxia. Amino acid incorporation into the proteins of all cell fractions was depressed stepwise with increasing degrees of hypoxia.

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